Determination of the substrate specificity of turnip mosaic virus NIa protease using a genetic method

The RNA genome of turnip mosaic potyvirus (TuMV) encodes a large polyprotein that is processed to mature proteins by virus-encoded proteases. The TuMV NIa protease is responsible for the cleavage of the polyprotein at seven different locations. These cleavage sites are defined by a conserved sequence motif Val-Xaa-His-Gln $, with the scissile bond located after Gln. To determine the sub-strate specificity of the NIa protease, amino acid sequences cleaved by the NIa protease were obtained from randomized sequence libraries using a screening method referred to as GASP (genetic assay for site-specific proteolysis). Based on stat-istical analysis of the obtained sequences, a con-sensus substrate sequence was deduced: Yaa-Val